Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on pulmonary tissue and on diabetic ulcer tissue, causing chronic antibiotic-tolerant infections. P. aeruginosa biofilms contain physiological heterogeneous subpopulations of cells, including dormant cells in the anaerobic or the nutrient poor biofilm microenvironments. Since most antibiotics target active cells, the dormant cells may survive the antimicrobial therapies, then repopulate the biofilms when treatments are discontinued. Longitudinal genomics studies of P. aeruginosa isolated from chronic pulmonary infections of cystic fibrosis (CF) patients support this mechanism of antibiotic tolerance, since clones of the original founder strains often reemerge within patients treated with antimicrobial therapies. In order to treat chronic infections, it will be necessary to prevent resuscitation of the dormant bacteria, in addition to killing the active cells. Here, we propose to identify the molecular targets that, when inhibited, impair P. aeruginosa resuscitation from starvation-induced dormancy. In prior work, we characterized the P. aeruginosa ribosomal hibernation promoting factor (HPF), which is required for protection of a minimum ribosome supply in starved P. aeruginosa cells, and therefore required for de novo protein synthesis of resuscitating cells. Protection of other macromolecules or cellular structures, in addition to ribosomes, is also likely required for dormant cells to resuscitate, since dormant cells are subject to aging, protein oxidation, and protein degradation. In preliminary studies, we identified additional mutant strains where gene disruptions prevent optimal resuscitation of P. aeruginosa from starvation. In the research here, we propose to continue to identify and characterize P. aeruginosa dormancy factors. Homologs to the factors identified here may be used to study dormancy in other bacteria that cause chronic infections. In this research, we will: (i) Perform a comprehensive transposon screen for mutations that affect the ability of P. aeruginosa to recover from starvation. The genes of the resulting mutant strains will be characterized for their effect on maintenance of macromolecular integrity during dormancy. (ii) Characterize physiological heterogeneity of the candidate mutant strains cultured in biofilms. Since dormant cells have greater tolerance to antibiotics than active cells, we will differentially label dormant and active cells, and determine if the gene disruptions affect the ability of antibiotic-treated dormant cells to resuscitate. Ultimately, we will identify new molecular targets that when inhibited may not have an effect on active cells, but that inhibit the dormant cells from resuscitation. Therapies that target these dormancy factors may then be used in combination with traditional antibiotic therapies to help prevent chronic and persistent infections.